PROJECT SUMMARY The classic model of G protein coupled receptor (GPCR) activation centers on ligand binding, G protein activation, and signal transduction via G protein-mediated signaling events. This paradigm has been called into question however, with the finding that some ligands ?including endogenous ligands and therapeutic agents? have a preference for beta-arrestin mediated pathways. However, the information flow from receptor activation to signaling cascades and the mechanism of beta-arrestin signaling are not well understood. This proposal will elucidate, at the molecular level, the fundamental mechanisms controlling ligand-specific induction of beta- arrestin signaling with two highly clinically relevant GPCRs, the cannabinoid 2 receptor (CB2R) and the mu opioid receptor (MOR). These human receptors bind the plant-derived cannabinoids and opioids leading to psychostimulant effects and reduction of pain. Precise control of receptor activation and signaling is critical to obtain only the desired therapeutic results, however, and not the undesired side effects such as tolerance, drug abuse and dependence. Substantial preliminary studies identified ligand-specific dwell times, i.e. the time receptors are clustered into clathrin coated pits with beta-arrestins before endocytosis, as a mechanism controlling beta-arrestin signaling. This trafficking event can be chemically and genetically modulated to selectively control beta-arrestin signaling, providing novel therapeutic strategies. This project will combine state-of-the-art live cell imaging technologies (total internal reflection fluorescence and spinning disk microscopies), and biochemical approaches to determine if ligand-specific dwell times are a general event controlling beta-arrestin signaling. Multiple ligands for these receptors will be investigated in heterologous systems and in cells endogenously expressing the receptors. Preliminary results in primary cultures strongly support our hypothesis that long dwell times correlate with beta-arrestin signaling. The aims are to: (1) examine endocytosis of the CBR2 and MOR at the single endocytic pit level, and (2) define the impact on cellular mechanisms of CB2R and MOR mediated beta-arrestin signaling, including whether endocytic dwell times can modulate these pathways. Results will provide a physiological role for the previously described variability in endocytic dwell times. These findings may be extended to future drug discovery efforts, including for other GPCRs, to rationally design therapeutic agents with specific outcomes in areas intractable via current technology.